卷烟烟气对细胞线粒体氧化损伤的研究

Oxidative damage of cellular mitochondria by cigarette smoke

目的研究卷烟烟气凝集物(cigarette smoke condensate,CSC)对人乳头状病毒永生化的人支气管上皮细胞(human papillomavirus-immortalized human bronchial epithelial cell line,BEP2D)线粒体的氧化损伤。方法采用分子探针2’,7’-二氯荧光黄双乙酸盐(2’,7’-dichlorofluorescein diacetate,DCFH-DA)和氢化乙啶(hydroethidine,HE)检测细胞内活性氧(reactive oxygen species,ROS)水平;用荧光标记物MCB(Monochlorobimane)、壬基吖啶橙(nonyl acridine orange,NAO)、四氯四乙基苯并咪唑基羰花青碘化物(5,5’,6,6’-tetrachloro-1,1’,3,3’-tetrethyl benzimidalyl carbocyanine iodide,JC-1)检测细胞内还原型谷胱甘肽(glutathione,GSH)及线粒体内膜心磷脂(cardiolipin,CL)、膜电位(mitochondrial membrane poten-tial,Δψm)。结果CSC作用BEP2D细胞后,细胞内ROS显著增加,GSH及线粒体CL、Δψm水平明显下降,并呈现较好的剂量效应关系。结论CSC可造成细胞线粒体氧化损伤。

Objective To investigate oxidative damage of mitochondria （CSC） in immortalized human bronchial epithelial cells （BEP2D）. Methods by cigarette smoke condensate BEP2D cells were treated with 0, 5, 10, 15, 20 μg/ml CSC respectively. 2＇ ,7＇-dichlorofluorescein （DCF） and ehidium bromide （EB）, the fluorescent products of the membrane-permeable dyes [ 2＇ , 7 ＇ -dichlorofluorescein diacetate （DCFH-DA） and Hydroethidine （HE） ] were used to monitor the intracellular production of reactive oxygen species （ROS） by Fluroskan Ascent FL. The cells were labeled by fluorescent dyes （ Monochlorobimane, nonyl acridine orange or 5,5＇ ,6,6＇ -tetrachloro-1,1 ＇ ,3,3＇ -tetrethyl benzimidalyl carbocyanine iodide respectively） and then intra- cellular reduced glutathione （GSH） and mitochondrial cardiolipin, mitochondrial membrane potential were determined by monitoring the fluorescence intensity. Results The ROS production in CSC treated BEP2D cells increased remarkably, and the level of GSH, cardiolipin and membrane potential decreased obviously, both in a good dose-effect relation. Conclusion CSC causes oxidative damage to mitochondria.